Polymorphism analysis by nucleic acid structure probing with structure-bridging oligonucleotides
First Claim
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1. A method for capturing bridging oligonucleotides, comprising:
- a) providing;
i) a first folded target having a nucleic acid sequence comprising first and second portions, said first and second portions each comprising one or more double stranded regions and one or more single stranded regions;
ii) a second folded target having a nucleic acid sequence comprising a first portion that is identical to said first portion of said first folded target and a second portion that differs from said second portion of said first folded target because of a variation in nucleic acid sequence relative to said first folded target, said first and second portions each comprising one or more double stranded regions and one or more single stranded regions;
iii) first and second bridging oligonucleotides said first bridging oligonucleotide complementary to said first portion of said first and second folded targets and said second bridging oligonucleotide complementary to said second portion of said first and second folded targets; and
iv) a solid support comprising first, second, third and fourth capture zones, each zone capable of capturing and immobilizing said first and second bridging oligonucleotides;
b) contacting said first folded target with said first bridging oligonucleotide under conditions such that said first bridging oligonucleotide binds to said first folded target to form a probe/folded target complex in a first mixture;
c) contacting said first folded target with said second bridging oligonucleotide under conditions such that said second bridging oligonucleotide binds to said first folded target to form a probe/folded target complex in a second mixture;
d) contacting said second folded target with said first bridging oligonucleotide to form a third mixture;
e) contacting said second folded target with said second bridging oligonucleotide to form fourth mixture; and
f) adding said first, second, third and fourth mixtures to said first, second, third and fourth capture zones of said solid support, respectively, under conditions such that said first and second bridging oligonucleotides are captured and immobilized.
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Abstract
The present invention relates to methods and compositions for analyzing nucleic acids. In particular, the present invention provides methods and compositions for the detection and characterization of nucleic acid sequences and sequence changes. The methods of the present invention permit the detection and/or identification of genetic polymorphism such as those associated with human disease and permit the identification of pathogens (e.g., viral and bacterial strain identification).
45 Citations
29 Claims
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1. A method for capturing bridging oligonucleotides, comprising:
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a) providing;
i) a first folded target having a nucleic acid sequence comprising first and second portions, said first and second portions each comprising one or more double stranded regions and one or more single stranded regions;
ii) a second folded target having a nucleic acid sequence comprising a first portion that is identical to said first portion of said first folded target and a second portion that differs from said second portion of said first folded target because of a variation in nucleic acid sequence relative to said first folded target, said first and second portions each comprising one or more double stranded regions and one or more single stranded regions;
iii) first and second bridging oligonucleotides said first bridging oligonucleotide complementary to said first portion of said first and second folded targets and said second bridging oligonucleotide complementary to said second portion of said first and second folded targets; and
iv) a solid support comprising first, second, third and fourth capture zones, each zone capable of capturing and immobilizing said first and second bridging oligonucleotides;
b) contacting said first folded target with said first bridging oligonucleotide under conditions such that said first bridging oligonucleotide binds to said first folded target to form a probe/folded target complex in a first mixture;
c) contacting said first folded target with said second bridging oligonucleotide under conditions such that said second bridging oligonucleotide binds to said first folded target to form a probe/folded target complex in a second mixture;
d) contacting said second folded target with said first bridging oligonucleotide to form a third mixture;
e) contacting said second folded target with said second bridging oligonucleotide to form fourth mixture; and
f) adding said first, second, third and fourth mixtures to said first, second, third and fourth capture zones of said solid support, respectively, under conditions such that said first and second bridging oligonucleotides are captured and immobilized. - View Dependent Claims (2, 3, 4, 5)
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6. A method for forming a probe/folded target complex, comprising:
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a) providing;
i) a first folded target having a nucleic acid sequence comprising first and second portions, wherein said first and second portions each comprise one or more double stranded regions and one or more single stranded regions;
ii) a second folded target having a nucleic acid sequence comprising a first portion that is identical to said first portion of said first folded target, and a second portion that differs from said second portion of said first folded target because of a variation in nucleic acid sequence relative to said first folded target, said first and second portions each comprising one or more double stranded regions and one or more single stranded regions;
iii) a solid support comprising first and second testing zones, each of said zones comprising immobilized first and second bridging oligonucleotides, said first bridging oligonucleotide complementary to said first portion of said first and second folded targets and second bridging oligonucleotide complementary to said second portion of said first and second folded targets; and
b) contacting said first and second folded targets with said solid support under conditions such that said first and second bridging oligonucleotides hybridize to said first folded target to form a probe/folded target complex. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13)
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14. A method of identifying a nucleotide sequence of a target nucleic acid based on its folded structure, comprising the steps of:
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a) providing;
i) two or more copies of a target nucleic acid suspected of having a particular nucleotide sequence, said sequence permitting formation of a folded structure comprising one or more single-stranded regions and further comprising two or more non-contiguous portions and one or more intervening regions, ii) a first oligonucleotide probe comprising a bridging oligonucleotide probe comprising at least two regions, each of said regions being complementary to a different one of at least two of said non-contiguous portions of said target nucleic acid, and iii) a second oligonucleotide probe selected from the group consisting of a non-bridging probe having a region of complementarity to said target nucleic acid, and a second bridging oligonucleotide comprising at least two regions, each of said regions being complementary to a different one of at least two of said non-contiguous portions of said target nucleic acid;
b) contacting at least one copy of said target nucleic acid with said first oligonucleotide probe to form a first probe/folded target complex;
c) contacting at least one copy of said target nucleic acid with said second oligonucleotide to form a second probe/folded target complex; and
d) measuring amounts of each of said first and said second probe/folded target complexes wherein the measured amount of one of said probe/folded target complexes as compared to the other probe/folded target complex is indicative of the sequence of the target nucleic acid. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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Specification